Detection of mutations of genes including a single nucleotide polymorphism (SNP) is widely conducted in prevention and treatment for all sorts of diseases. For example, many mutations are observed in genes of cancer cells and it is known that these mutations are involved in malignant transformation of cells. Therefore, detection of mutations of genes in cells makes it possible to detect stage progression of malignant transformation, and it is considered as very useful tool in treatment. Further, it is reported that there are mutations showing drug resistance in genes of cancer cells on medication. Since detection of these mutations makes it possible to judge effectiveness of drugs for each patient, further appropriate treatment can be conducted. For example, although a treatment with medication of an anticancer agent “imatinib” is widely conducted for chronic myelogenous leukemia (CML), it is considered that a mutation (for example, T315I) of the bcr-abl gene affects drug resistance. Since detection of mutations of genes is useful for early detection and treatment in a clinical field, high reliability of the detection is required.
As the method for detecting mutations of genes, a direct sequencing method, an ASP (allele specific primer)-PCR (polymerase chain reaction) method (Patent Document 1), and a Tm (melting temperature) analysis method (Non Patent Document 1) are generally known. The direct sequencing method is a method in which a region including an objective base site to be detected is amplified and a base sequence of the thus obtained amplification product is analyzed. The ASP-PCR method is a method in which PCR is conducted using a primer that is complementary to a region including an objective base site to be detected and has a base complementary to a base of the objective base site to be detected at the 3′ end region and a mutation is judged on the presence or absence of amplification. According to this method, for example, when a primer that is complementary to a sequence in which the objective base site to be detected is set as a mutant-type base (hereinafter, also referred to as a “mutant sequence”) is used, a mutation can be checked by the detection of the amplification. Further, when a primer that is complementary to a sequence in which the objective base site to be detected is set as a normal-type base (hereinafter, also referred to as a “normal sequence”) is used, a non-mutation (i.e., normal) can be checked by the detection of the amplification. In the Tm analysis, first, a region including the objective base site to be detected is amplified and a hybrid (double-stranded DNA) between the thus obtained amplification product and a probe that is complementary to the mutant sequence is formed. Then, this hybridization product is heat-treated, dissociation (melting) of the hybrid accompanying the temperature rise is detected by measuring signals such as an absorbance and the like, and thus a Tm value is determined. Accordingly, the presence or absence of the mutation is judged. The more the respective chains of the hybridization product match, the higher the Tm value, and the less the respective chains of the hybridization product match, the lower the Tm value. Therefore, when the Tm value (reference value for assessment) is determined beforehand with respect to the hybridization product between the mutant sequence and a probe that is complementary thereto, and this reference value is compared to the determined Tm value (measurement value), the following judgment can be made. When the measurement value is identical to the reference value, it is considered as perfect match, that is, it can be judged that a mutation is present in the objective base site to be detected. On the other hand, when the measurement value is lower than the reference value, it is considered as mismatching, that is, it can be judged that the objective base site to be detected is normal, i.e., no mutation is present.
However, the direct sequencing method has low sensitivity and the operation thereof requires considerable time and effort. The ASP-PCR method has high sensitivity, but has low specificity. That is, when a primer that is complementary to the mutant sequence is used, there is a possibility that amplification is observed even though no mutation is present and that it is judged as false-positive. Further, in the ASP-PCR method, only one of a primer that is complementary to the mutant sequence and a primer that is complementary to the normal sequence can be used in one reaction system. Therefore, in order to check whether the objective base site to be detected is normal or mutant, PCR should be conducted with reference to two reaction systems that is a reaction system using a primer that is complementary to the mutant sequence and a reaction system using a primer that is complementary to the normal sequence. Since the ASP-PCR method uses two reaction systems, the operation thereof requires time and effort and it is costly. Further, there is a problem that the judgment with sufficient reliability is difficult even though two reaction systems are used. That is, with respect to the two reaction systems, when no amplification is observed in the reaction system using a primer that is complementary to the normal sequence and the amplification is observed in the reaction system using a primer that is complementary to the mutant sequence, it can be judged as positive with no doubt. However, when the amplification is observed in the both reaction systems, it is still difficult to judge whether the objective base site to be detected is normal or mutant. On the other hand, since the Tm analysis method has high specificity, the problem of a false-positive can be avoided. Further, it can be judged whether the objective base site to be detected is normal or mutant in one reaction system. However, there is a problem that the Tm analysis method has insufficient sensitivity.
Particularly, when a mutation relating to a cancer is detected as described above, both cells in which objective genes are mutated and cells in which objective genes are normal are present in specimens collected from patients. Therefore, it is required to detect the presence or absence of the mutation accurately with respect to, for example, biological samples containing large amounts of normal genes and small amounts of mutant genes.    Patent Document 1: JP 2853864 B    Non Patent Document 1: Analytical Biochemistry 290, 89-97 (2001)